The p53 tumor suppressor and master regulator is central to human DNA repair and damage checkpoints. It also has key roles in many aspects of human biology. Importantly, most cancers are altered for p53 function. The Chromosome Stability Group (CSG) has discovered a greatly expanded universe of p53 targets and human diversity as well as variations in responses to specific stresses such as UV and anticancer agents. The CSG approaches have provided functional analysis of cancer-associated mutants from tumors and revealed functional interactions with the estrogen receptor master regulatory network. FUNCTIONAL P53 TARGET SEQUENCES AND AN EXPANDING UNIVERSE. The DNA binding and transactivation of the master regulator p53 is critical to tumor suppressor activity in response to cellular and environmental stresses. Across hundreds of targeted genes in the p53 stress response network there is considerable variation in p53 dependent expression leading to differences in p53-mediated biological consequences. Much of that is due to target RE sequence. We focus on the functionality of REs, i.e., the ability of REs to support transactivation by p53. The functionality is determined by many factors, the most important being sequence and level of p53. To directly assess transactivation responsiveness of human REs, we developed promoter systems in budding yeast for variable p53 expression (i.e., rheostatable). This has been used to establish the functional evolution of REs across species (discussed below) and has been summarized in our piano model that describes functional variability within a transcriptional network. We have translated many of the findings in yeast to p53 in human cells in culture and ex vivo. Recently, we established that increases in spacer length of only a few bases in a target RE greatly reduces functionality in terms of p53 transactivation. In collaboration with Gilbert Schoenfelder (Berlin), we identified a novel mechanism of p53 transcriptional control of genes through studies of a SNP located in the promoter of the Flt1 gene. This C to T SNP results in generation of a perfect p53 half site RE and is present in 5-10% of the population. Our findings provided the first report of a 1/2 site functional for transactivation in the p53 network. Endogenous p53-dependent induction of FLT-1 mRNA was only observed in cell lines containing the FLT1-T allele in response to several DNA damaging agents such as doxorubicin, UV and ionizing radiation. These results established that p53 can differentially stimulate transcription at a polymorphic variant of the FLT1 promoter and placed the VEGF system directly in the p53 stress-response transcriptional network via FLT1 in a significant number of people. We extended the findings with 1/2 sites and recently examined transactivation capacity towards a panel of 15 p53 1/2 sites in p53-null human osterosarcoma cells (SaOS2) transiently co-transfected with a WT p53 vector. Some 1/2 site REs supported p53 transactivation to levels similar to a weakly responding full site RE from the apoptosis-related AIP gene. This finding greatly expands the potential p53 master regulatory network. INTERACTION OF p53 AND ESTROGEN RECEPTOR MASTER REGULATORY NETWORKS. Further analysis using several FLT1-T promoter::reporters in human cells revealed that the 1/2 site only supported modest p53 transactivation. We identified an estrogen receptor (ERE) 1/2 site RE that was required for enhanced p53 induced transactivation at the p53 RE located 225nt downstream. Transcriptional interaction between the two 1/2 sites in cis, as well as another ERE site, was demonstrated by cooperative stimulation when both ER alpha and p53 were expressed. We have now established a new dimension to the p53 master regulatory network where p53-mediated transcription from a 1/2 site RE can be determined by ER binding at one or more cis-acting EREs in a manner dependent on ER level ER, the type of ligand and specific p53-inducing agent. Recently, we addressed the generality of synergistic transactivation by p53 and ER. The 1/2 site in the FLT1 promoter was replaced with various 1/2 sites, as well as canonical weak and strong human p53 REs.p53 transactivation was greatly enhanced by ligand-activated ER acting in cis. Furthermore, enhanced transactivation extends to several cancer-associated p53 mutants with altered function, suggesting ER-dependent mutant p53 activity for at least some REs and possibilities for reactivation of cancer mutants. We propose a general synergistic relationship between the p53 family and ER master regulators in transactivation of p53 target canonical and noncanonical REs which might be poorly responsive to p53 on their own. We are developing a functional matrix as a tool for genome wide searches for putative p53 target genes via noncanonical sites and augmenting transcriptional factors. For example, in a limited human genome search to identify motifs containing both strong p53 noncanonical RE sequences (including decamers with a CATG core) and associated ER responsive sequences (FLT1-like motifs), we identified the RAP80 gene and Toll-like receptors (TLRs) that determine innate immunity. The identification of potential p53 target REs associated with the Toll-like receptors has led to exciting new findings. Among the ten human TLRs, nine had canonical and noncanonical p53 REs that may be functional. In agreement with bioinformatics, genotoxic activation of p53 or upregulation of p53 protein level is able to induce expression of many TLRs in various human cancer cell lines and ex vivo in peripheral blood cells. CANCER-ASSOCIATED P53 MUTANTS. Using yeast-based and human cell systems, we show that functional p53 mutations can lead to considerable diversity in the spectrum of responses from different REs that include 1) decrease/loss-of-function;2) subtle changes;3) altered specificity;and 4) super-transactivation all of which lead to variation in biological responses. Recently we extended our findings regarding transactivation by p53 mutants at full site REs to noncanonical p53 half sites. Most of the functional mutants (8/9) were able to function at 1/2 sites. We extended our analysis of p53 mutants in yeast based systems to a clinical study of breast cancer patients undergoing neoadjuvant treatment prior to surgery. Several transcriptionally active p53 missence mutants identified in breast as well as adrenal gland cancers (ACC) had subtle defects in transcription at REs that can only be revealed at low expression levels achievable in the yeast-based system. The indications so far are that nonfunctional as compared with functional missense mutants were more likely to exhibit stage III progression at diagnosis, high grade cancers and to relapse with cancers at distant sites. There also may be ethnic differences. P53 NETWORK EVOLUTION. We investigated evolution of REs in terms of responsiveness to p53. Individual REs exhibited marked differences in potential transactivation as well as widespread turnover of functional binding sites during p53 network evolution. Only 1/3 of the REs found in humans are predicted to be functionally conserved in rodents. Surprisingly, the p53 responsiveness of the DNA repair/metabolism set of genes in humans has evolved separately from mice suggesting differences in responsiveness to cancer-inducing agents between mice and humans. Of 15 p53 targeted human REs from 12 genes affecting DNA metabolic activities none are p53 responsive in rodents. Interestingly, we found functional conservation of weakly responding REs including 1/2 sites. Among validated p53 REs conserved between rodents and humans, one third were comprised of 1/2 or 3/4 sites, each with a perfect consensus 1/2 site suggesting a selective advantage in retaining weak p53 REs.